The primary goal of this proposal is to gain a better understanding of the regulation of GLUT1 (SLC2A1) activity. GLUT1 activity is relevant to diabetes and metabolic syndrome where glucose uptake is compromised, and to cancer, where GLUT1 activity is enhanced. Understanding how GLUT1 activity is regulated may provide insights into avenues for therapeutic intervention in these diseases. Furthermore, the project is highly accessible to undergraduate students and has clear relevance to their post-graduate and medical education. Prior research has demonstrated that GLUT1 can be acutely activated by a wide variety of metabolic stressors and thiol-reactive compounds, which implicates the importance of cysteine residues in GLUT1 activity. We will explore acute changes of glucose uptake in two cell lines that exclusively express GLUT1. L929 cells have low GLUT1 abundance are sensitive to thiol-reactive GLUT1 activators, while HCLE (human corneal limbal epithelial) have higher levels of membrane GLUT1 fail to further increase glucose uptake in response to these compounds, but rather, uptake actually slows, suggesting that their complement of GLUT1 transporters already exist in the higher activity conformation. These two cell lines will allow us to study changes in GLUT1 activity bidirectionally; activation in L929 and deactivation in HCLE cells. We hypothesize that GLUT1 activity is positively regulated by multimerization, which stabilizes a high-activity conformation and is a function of the membrane concentration and lateral mobility of GLUT1. GLUT1 tetramers are more active than the monomeric form but it has not yet been demonstrated that that acute changes in GLUT1 activity correlate with changes in GLUT1 quaternary structure. We will address our central hypothesis with three specific aims. Aim 1: We will examine the relationship between the membrane concentration and activity of GLUT1 in the presence or absence of acute activators. Expression of GLUT1 in L929 will be increased using a tetracycline- inducible system, while titrating in GLUT1-specific siRNAs will decrease expression of GLUT1 in HCLE cells. We expect that as GLUT1 increases there will be a loss of responsiveness to activation. Aim 2: We will determine if acute activation of GLUT1 induces multimerization of GLUT1. We will examine endogenous GLUT1 molecular weights pre- and post-activation by blue native gel electrophoresis, size exclusion chromatography and membrane crosslinking approaches. We will also examine the changes in oligomerization of exogenous GLUT1 by using BRET, FRAP, and co-immunoprecipitation techniques. We expect that activation of GLUT1 will increase aggregation of GLUT1. Aim 3: We will determine if GLUT1 activation alters lateral diffusion via changes in GLUT1 tethering. We will utilize a novel labeling and fractionation procedure that allows us to detect GLUT1 tethered to F-actin. We will also use FRAP to determine if there is a change in the mobile fraction after acute activation. We expect that activation will promote lateral mobility of GLUT1 by decreasing tethering to F-actin and allow for increased aggregation of GLUT1.